Abstract
The potential of human mesenchymal stromal/stem cells (MSCs) including oral stem cells (OSCs) as a cell source to derive functional neurons has been inconclusive. Here we tested a number of human OSCs for their neurogenic potential compared to non-OSCs and employed various neurogenic induction methods. OSCs including dental pulp stem cells (DPSCs), gingiva-derived mesenchymal stem cells (GMSCs), stem cells from apical papilla and non-OSCs including bone marrow MSCs (BMMSCs), foreskin fibroblasts and dermal fibroblasts using non-neurosphere-mediated or neurosphere-mediated methods to guide them toward neuronal lineages. Cells were subjected to RT-qPCR, immunocytofluorescence to detect the expression of neurogenic genes or electrophysiological analysis at final stage of maturation. We found that induced DPSCs and GMSCs overall appeared to be more neurogenic compared to other cells either morphologically or levels of neurogenic gene expression. Nonetheless, of all the neural induction methods employed, only one neurosphere-mediated method yielded electrophysiological properties of functional neurons. Under this method, cells expressed increased neural stem cell markers, nestin and SOX1, in the first phase of differentiation. Neuronal-like cells expressed βIII-tubulin, CNPase, GFAP, MAP-2, NFM, pan-Nav, GAD67, Nav1.6, NF1, NSE, PSD95, and synapsin after the second phase of differentiation to maturity. Electrophysiological experiments revealed that 8.3% of DPSC-derived neuronal cells and 21.2% of GMSC-derived neuronal cells displayed action potential, although no spontaneous excitatory/inhibitory postsynaptic action potential was observed. We conclude that DPSCs and GMSCs have the potential to become neuronal cells in vitro, therefore, these cells may be used as a source for neural regeneration.
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Abbreviations
- AP :
-
Action potential
- BM :
-
Bone marrow
- BSA :
-
Bovine serum albumin
- DAPI :
-
4′,6-diamidino-2-phenylindole dihydrochloride
- DFs :
-
Human dermal fibroblasts
- DPSCs :
-
Dental pulp stem cells
- FFs :
-
Human foreskin fibroblasts
- GMSCs :
-
Gingiva-derived mesenchymal stem cells
- hESC :
-
Human embryonic stem cell
- iPSCs :
-
Induced pluripotent stem cells
- MSCs :
-
Mesenchymal Stromal/Stem Cells
- NDM :
-
Neural differentiation medium
- NMM :
-
Neurogenic maturation medium
- NSC :
-
Neural stem/progenitor cell
- OSCs :
-
Oral stem cells
- PD :
-
Population doubling
- qPCR :
-
Quantitative real-time polymerase chain reaction
- SCAP :
-
Stem cell of apical papilla
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Acknowledgments
The authors thank Dr. Kristen O’Connell (UTHSC) for her assistance during the early developmental stage of the project; Dr. Rebeca Caires Mugarra (UTHSC) for initial assistance in the patch-clamp studies; and UTHSC Biostatistic BERD Consulting Program for statistical support.
Funding
This work was supported in part by grants from the National Institutes of Health R01 DE019156 (G.T.-J.H.), AHA 15SDG25700146 (J.F.C.), National Institutes of Health R01 GM125629–01 (J.F.C-M) and a Research Fund from the University of Tennessee Health Science Center (G.T.-J.H.).
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DL designed and performed the experimental work, acquired, assembled, analyzed the data, and drafted/revised the manuscript. XYZ initiated the project and along with IEA, LOR, ZY, and AIL performed some of the experimental work, assembled, analyzed the data and drafted/revised part of the manuscript. JFCM supervised some of the experimental work, assembled, analyzed the data and drafted/revised part of the manuscript. GTJH conceived, designed, performed experimental works and supervised the overall project, analyzed and interpreted the data and finalized the manuscript. All authors have read and approved the manuscript for publication.
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To be discarded extracted teeth were collected from Clinics at Boston University (BU) and University of Tennessee Health Science Center (UTHSC) based on exempt protocols approved by the respective Medical Institutional Review Board (BU: #H-28882; and UTHSC:#12–01937-XM).
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The authors declare that they have no competing interests.
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Supplemental Fig. 1
RT-PCR analysis of the expression of neural markers at day 0 (before induction). Donor information is the same as that in Fig. 2. (PNG 189 kb)
Supplemental Fig. 2
qPCR analysis of non-neurosphere mediated neurogenic induction (NDM-C). Representative data measured in triplicate showing various neurogenic gene expression in OSCs and BMMSCs at 0, 7, 21 and 35 days following induction. OSCs: DPSCs (donor: 18-yr female, passage 3); SCAP (donor: 17 yr. female, p3); GMSCs. (donor: 53 yr. male, p4). BMMSCs (20 yr. male, p4). (Donors were different from those presented in Fig. 2) (PNG 1181 kb)
Supplemental Fig. 3
Immunofluorescence analysis after neurogenic induction (NDM-C) for 21–35 days. Top panel: Oral stem cells including DPSCs, SCAP and GMSCs (GMSCs from A and B two donors). Bottom panel: Non-OSCs including BMMSCs, FFs and DFs. Genes tested included neural genes nestin, βIII-tubulin, NFM, Nav1.6 and CNPase. Scale bar: DPSCs, all 50 μm; SCAP, all 100 μm; GMSCs and BMMSCs, all 50 μm; DF, nestin, 100 μm; NFM, 200 μm; Nav1.6, 50 μm. (PNG 24206 kb)
Supplemental Fig. 4
Neurosphere formation and differentiation of DPSCs (Method-1). Upper panel: (A, B) DPSC-derived neurospheres after 3 days cultured in NDM-1 stage-1. (C) Neural differentiation of DPSC-derived neurosphere on day 2 (NDM-1 stage-2). (D) After 6 days of neural differentiation (NDM-1 stage-2), fibroblast-like cells became confluent in the central area of neurospheres. Lower panel: A few cells with long processes could be seen in the peripheral area of neurospheres, while the cell bodies were not spheroidal. Immunofluorescence staining showing βIII-tubulin-positive cells (red). DAPI: nuclear stain. Mouse IgG serves as the negative control. Scale bar: all the scale bars are 100 μm, except in (D), 500 μm. (PNG 2241 kb)
Supplementary Fig. 5
Neurogenesis of human DPSC neurospheres on low-attachment and adherent plates under low and normal oxygen conditions (Method-2). (A) Formation of primary (72 h) DPSC neurospheres under normoxic and hypoxic conditions at stage-1 (non-adherent images on left). Representative images of neurospheres cultured on adherent plates after 7 days (middle) and 12 days (right) with neurodifferentiation medium (stage-2). Similar cell morphological characteristics are observed in both normoxic and hypoxic plates. Scale bars: 100 μm. (B) Immunofluorescence analysis of neural markers. Cultured DPSC neurospheres were induced with neurodifferentiation media for 12 days under normoxic or hypoxic conditions. DAPI: nuclear stain; βIII-tubulin: neuronal-specific marker (green); GFAP: astrocyte-specific marker (red); Merged image with all three fields. Scale bars: 100 μm. (PNG 5173 kb)
Supplemental Fig. 6
Neurosphere-mediated neuronogenesis (Method-3) of human GMSCs. Neurospheres formed under neural induction stage for 6–8 days as the spheres increased in size over time (stage-1). Representative images of late phase (day 6). After which, spheres were seeded onto poly-l-ornithine/laminin coated glass coverslips or culture wells and stimulated under neural maturation medium for ~4 weeks (stage-2). The cells gradually showed spherical cell body and axon-like extensions over time. Representative images showing early phase (day 4) and late phase (day 28). Control: non-stimulated. Scale bars: Top panel, 500 μm (left 2 images,) 100 μm (right 2 images). Bottom panel, 100 μm (left 2 images), 50 μm (right 2 images). (PNG 4084 kb)
Supplemental Fig. 7
Neurosphere-mediated neuronogenesis (Method-3) of GMSCs. After stage-1 neurosphere step, the spheres were seeded onto wells of a 24-well plate under stage-2 NMM. Selected locations of the attached spheres were imaged consecutively starting at day 1 throughout the maturation period. The images were combined into a video format. Two locations each contains a single cell were traced (red and green boxes) backward from day 28 to day 1. From day 28 to 17, the same cell was recognized and traced. Days before the 17th day, the same cell was not easily recognized due to migration or crowdedness of cells. (MP4 19,552 kb)
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Li, D., Zou, XY., El-Ayachi, I. et al. Human Dental Pulp Stem Cells and Gingival Mesenchymal Stem Cells Display Action Potential Capacity In Vitro after Neuronogenic Differentiation. Stem Cell Rev and Rep 15, 67–81 (2019). https://doi.org/10.1007/s12015-018-9854-5
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DOI: https://doi.org/10.1007/s12015-018-9854-5